Use of il-17a as a target in the preparation of a medicament for screening, treating or preventing cardiac arrest

Abstract

The application provides application of IL-17A as a target point in preparation of a drug for screening treatment or prevention of cardiac arrest. The application also provides application of IL-17A as a target point in preparation of a monoclonal antibody drug for screening treatment or prevention of cardiac arrest. The application also provides application of an anti-IL-17A monoclonal antibody in preparation of a drug for improving post-cardiac arrest syndrome. The application provides application of an anti-IL-17A monoclonal antibody in preparation of a drug for early intervention of cardiac arrest. The application also provides application of an anti-IL-17A monoclonal antibody in preparation of a drug for improving myocardial injury, cardiac dysfunction, brain injury or neurological dysfunction caused by cardiac arrest. The application also provides application of a reagent for detecting IL-17A level in serum in preparation of a kit for detecting a myocardial infarction combined with cardiac arrest patient. The application proves by experiments that IL-17 related pathways in heart and brain tissues are significantly activated after cardiac arrest, and IL-17A in serum is significantly increased, and early antagonism of IL-17A by using an IL-17A monoclonal antibody after resuscitation can improve myocardial injury, cardiac dysfunction (systolic and diastolic dysfunction), brain tissue injury or neurological dysfunction after cardiac arrest, and improve survival rate after cardiac arrest.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine and relates to a therapeutic target and cardiovascular drugs, particularly the application of IL-17A as a target in the preparation and screening of drugs for the treatment or prevention of cardiac arrest, and the use of anti-IL-17A monoclonal antibodies in the preparation of drugs to improve the prognosis of cardiac arrest. Background Technology

[0002] Statistics show that an average of 550,000 people in China experience cardiac arrest (CA) each year, the highest number in the world. Although the widely implemented cardiopulmonary resuscitation (CPR) and automated external defibrillators (AEDs) can save lives to some extent, the survival rate remains less than 10%. These alarming figures demonstrate that our understanding of this disease is still extremely limited; therefore, actively seeking new research breakthroughs is of paramount importance.

[0003] Post-cardiac arrest syndrome (PCAS) is a key factor leading to early death in patients, comprising four interacting components: systemic ischemia / reperfusion response, brain injury, myocardial dysfunction, and persistent precipitating factors of cardiac arrest. During cardiac arrest, the patient is in a state of systemic ischemia, and after the restoration of spontaneous circulation, reperfusion injury occurs. Early ischemia and reperfusion injury trigger an inflammatory cascade response, leading to a systemic inflammatory cascade or sepsis-like syndrome, and subsequent multi-organ dysfunction. Therefore, early intervention in the inflammatory response after cardiac arrest to break the PCAS interactions is of significant clinical importance for the prognosis of cardiac arrest. However, to date, interventions for cardiac arrest mainly include targeted temperature management and maintaining hemodynamic homeostasis; there is still no specific drug treatment for the early inflammatory cascade phase after cardiac arrest.

[0004] Interleukin-17A (IL-17A), produced by cells such as helper T17 (Th17) cells, γδT cells, and natural killer T (NKT) cells, has been shown to play a crucial role in host defense, allergic diseases, and autoimmune diseases. As a pro-inflammatory cytokine, IL-17A can induce the expression of chemokines and promote neutrophil infiltration into target organs. In recent years, evidence has shown that IL-17A can directly mediate cardiomyocyte apoptosis in cardiac ischemia / reperfusion injury and exacerbate ischemic brain injury. Monoclonal antibodies against IL-17A can improve the prognosis of stroke. However, the role of IL-17A in cardiac arrest and whether intervention with IL-17A can affect the prognostic outcome of cardiac arrest remain not fully understood.

[0005] Previous studies have shown that serum inflammatory cytokines such as IL-6 and IL-23 are important biological indicators for evaluating the prognosis of cardiac arrest, and early elevations in IL-6 and IL-23 are significantly associated with poor prognosis in cardiac arrest. However, a single-center clinical cohort study of out-of-hospital cardiac arrest patients showed that while infusion of the IL-6 receptor antagonist tocilizumab could improve myocardial injury after cardiac arrest, it did not significantly affect brain injury or survival rate after cardiac arrest.

[15] Furthermore, there is currently no evidence to show the effect of antagonizing IL-23 on the prognosis of cardiac arrest. These results indicate that the biological markers of the early inflammatory cascade after cardiac arrest that have been identified at this stage are difficult to use as intervention targets, or are tissue-specific, have poor universality, and are not applicable to the treatment of PCAS.

[0006] Secukinumab is a recombinant, high-affinity, fully humanized anti-IL-17A monoclonal immunoglobulin G1K antibody, approved by the European Union and the US FDA in January 2015 for the treatment of moderate to severe plaque psoriasis. This drug selectively binds to IL-17A, thereby significantly blocking the inflammatory cascade response in psoriasis patients, with minimal impact on other immune functions. Summary of the Invention

[0007] To address the aforementioned technical problems in the prior art, this invention provides the application of IL-17A as a target in the preparation and screening of drugs for the treatment or prevention of cardiac arrest. This application of IL-17A as a target in the preparation and screening of drugs for the treatment or prevention of cardiac arrest aims to solve the technical problem that existing drugs have poor therapeutic effects on the early inflammatory cascade stage after cardiac arrest.

[0008] This invention provides the application of IL-17A as a target in the preparation and screening of drugs for the treatment or prevention of cardiac arrest.

[0009] This invention also provides the application of IL-17A as a target in the preparation of monoclonal antibody drugs for screening the treatment or prevention of cardiac arrest.

[0010] The present invention also provides the use of anti-IL-17A monoclonal antibody in the preparation of medicaments for improving post-cardiac arrest syndrome.

[0011] This invention also provides the use of anti-IL-17A monoclonal antibodies in the preparation of medicaments for early intervention in cardiac arrest.

[0012] The present invention also provides the use of anti-IL-17A monoclonal antibody in the preparation of medicaments for improving myocardial injury, cardiac dysfunction (systolic and diastolic dysfunction), brain tissue injury or neurological dysfunction caused by cardiac arrest.

[0013] Furthermore, the anti-IL-17A monoclonal antibody is secukinumab.

[0014] The present invention also provides the use of reagents for detecting serum IL-17A levels in the preparation of kits for detecting patients with myocardial infarction complicated by cardiac arrest.

[0015] This invention demonstrates through experiments that IL-17-related pathways are significantly activated in cardiac and brain tissues after cardiac arrest, and IL-17A is significantly upregulated in serum. Antagonizing IL-17A with IL-17A monoclonal antibodies in the early post-resuscitation period can improve myocardial injury, cardiac dysfunction (systolic and diastolic dysfunction), brain tissue injury, or neurological dysfunction after cardiac arrest, and improve the survival rate after cardiac arrest.

[0016] This invention provides a novel application of IL-17A, a cytokine, as a core target of the early multi-organ inflammatory cascade response following cardiac arrest resuscitation, in the treatment or prevention of cardiac arrest. Through animal experiments and clinical specimen observations, this invention elucidates the feasibility of using IL-17A in the development of drugs for the early intervention and treatment of cardiac arrest, a critical clinical condition.

[0017] Compared with existing technologies, the technical effects of this invention are positive and significant. This invention, through animal and clinical data, demonstrates for the first time the core role of IL-17A in influencing the prognosis of cardiac arrest. Furthermore, early antagonism of IL-17A using monoclonal neutralizing antibodies can significantly improve inflammation and dysfunction in multiple organ systems after cardiac arrest resuscitation, providing positive guidance for clinical drug intervention after cardiac arrest and offering a new strategy for the prevention and treatment of cardiac arrest in clinical practice. Attached Figure Description

[0018] Figure 1 The results showed that cardiac arrest induced a myocardial inflammatory response, as revealed by cardiac tissue transcriptomics.

[0019] Figure 2 The study revealed that brain tissue transcriptomics showed cardiac arrest induces an inflammatory response in the brain.

[0020] Figure 3 The changes in the expression of the IL-17 family in serum after cardiac arrest were shown.

[0021] Figure 4 Early antagonism of IL-17A was shown to improve myocardial inflammatory response and cardiac dysfunction after cardiac arrest.

[0022] Figure 5 Early antagonism of IL-17A was shown to improve brain tissue inflammatory response, neurological dysfunction, and survival rate after cardiac arrest. Detailed Implementation

[0023] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. These preferred embodiments are intended to describe and explain the present invention, but not to limit it.

[0024] Experimental methods not specified in the examples are generally performed under conventional conditions, such as those described in textbooks and experimental guides, or as recommended by the manufacturer.

[0025] Example 1: Transcriptomic analysis of myocardial tissue after cardiac arrest.

[0026] 1. Establishment of a mouse model of cardiac arrest:

[0027] ① Anesthesia: 6-8 week old C57BL / 6 mice (purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were fasted overnight and anesthetized using isoflurane.

[0028] ② Endotracheal intubation: After anesthesia, the mouse was fixed in a supine position and connected to an electrocardiogram telemetry system. The neck skin was cleaned and disinfected, and the muscles and connective tissue around the trachea were quickly separated after being cut with scissors to fully expose the trachea. An appropriately sized endotracheal tube was inserted through the mouse's mouth and smoothly advanced into the airway. After securing the tube, it was connected to a small animal ventilator for ventilation (tidal volume: 200 μL, respiratory rate: 150 breaths / min) and isoflurane anesthesia was maintained.

[0029] ③ Jugular vein catheterization: The right jugular vein of the mouse was freed, and the distal end was ligated with silk suture, while the proximal end was suspended with silk suture to block blood flow. A small incision was made in the middle of the blood vessel using microscissors, and the indwelling venous catheter was inserted through the incision and secured with silk suture. A small amount of physiological saline was injected to flush out the blood flowing back into the catheter to prevent thrombosis.

[0030] 2. Cardiac arrest induction and cardiopulmonary resuscitation

[0031] ① Cardiac arrest: A high concentration of KCl solution (0.08 mg / g) was rapidly injected into the jugular vein via a cannula, and the electrocardiogram changes were monitored in real time until the heart lost electrical activity. The duration of cardiac arrest was 9 minutes.

[0032] ② Cardiopulmonary resuscitation (CPR): CPR was initiated 9 minutes after cardiac arrest, using a combination of mechanical ventilation and mechanical chest compressions. The mechanical ventilation parameters were as described above, and the chest compression rate was approximately 350 compressions / min. During resuscitation, epinephrine hydrochloride was administered three times via the jugular vein (at the start of resuscitation, 1 minute after resuscitation, and 2 minutes after resuscitation) to stimulate the heart to regain spontaneous heart rate. CPR was stopped when the mouse regained spontaneous circulation or when CPR lasted for 5 minutes. The restoration of spontaneous circulation was considered complete when any of the following indicators were present: 1) Electrocardiogram showing a restored spontaneous heartbeat with a rate > 200 beats / min; 2) Mean arterial pressure > 40 mmHg.

[0033] 3. Postoperative management

[0034] After the mice regained spontaneous circulation, the arterial and venous catheters were removed, and the neck skin was sutured. The mice were continuously observed for changes in electrocardiogram and respiratory status. After 30 minutes, they were moved to a 32°C constant temperature heating platform. During this period, glucose saline was injected intraperitoneally to replenish lost electrolytes and water.

[0035] Heart tissues were collected from the cardiac arrest group (CA / CPR) and the sham operation group (Sham) 3 hours post-operation. Total RNA was extracted and transcriptome sequencing was performed. The results showed that cardiac arrest significantly induced a myocardial inflammatory response, with the IL-17 signaling pathway showing the most significant alterations (ranked first). Figure 1 AC), among which chemokine-related genes are significantly upregulated after cardiac arrest. Figure 1 D).

[0036] Figure A: PCA analysis revealed significant differences in gene expression at the transcriptomic level between the CA / CPR group and the Sham group of myocardial tissue.

[0037] Figure B: The volcano plot reveals the differential gene up- and down-regulation in the myocardial tissue of the CA / CPR group and the Sham group.

[0038] Figure C: KEGG pathway enrichment analysis of differentially expressed genes in myocardial tissue showed that the differentially expressed pathways before and after cardiac arrest were mainly related to the inflammatory response, among which the IL-17 signaling pathway showed the most significant changes.

[0039] Figure D: The heatmap shows the changes in IL-17 pathway gene expression levels before and after cardiac arrest.

[0040] Example 2

[0041] Transcriptomic analysis of brain tissue after cardiac arrest.

[0042] A mouse model of cardiac arrest was established according to the protocol in Example 1. Brain tissue was collected from the cardiac arrest group (CA / CPR) and the sham-operated group (Sham) 3 hours after surgery. Total RNA was extracted and transcriptome sequencing was performed. The results showed that cardiac arrest could significantly induce an inflammatory response in brain tissue, with alterations in the IL-17 signaling pathway ranking second. Figure 2 AC), among which chemokine-related genes are significantly upregulated after cardiac arrest. Figure 2 D).

[0043] Figure A: PCA analysis revealed significant differences in gene expression at the transcriptomic level between the brain tissues of the CA / CPR group and the Sham group.

[0044] Figure B: The volcano plot reveals the differential gene up- and down-regulation in the brain tissues of the CA / CPR and Sham groups.

[0045] Figure C: KEGG pathway enrichment analysis of differentially expressed genes in brain tissue showed that the differentially expressed pathways before and after cardiac arrest were mainly related to inflammatory responses, with the IL-17 signaling pathway being the second most altered.

[0046] Figure D: The heatmap shows the changes in IL-17 pathway gene expression levels before and after cardiac arrest.

[0047] Example 3

[0048] Detection of serum IL-17 family expression levels after cardiac arrest.

[0049] A mouse model of cardiac arrest was established according to the protocol in Example 1. Blood samples were collected from the cardiac arrest group (CA / CPR) and the sham operation group (Sham) 3 hours post-operation. Additionally, blood samples were collected from clinical patients with myocardial infarction complicated by CA and from patients with myocardial infarction without CA. All collected blood samples were left at room temperature for 1 hour to allow for complete coagulation, centrifuged at 3000 rpm at 4°C for 10 minutes, and serum was collected and aliquoted into 1.5 mL EP tubes to prevent repeated freeze-thaw cycles for subsequent detection of IL-17 family serum levels. By detecting the expression changes of all IL-17 family members (IL-17A, IL-17B, IL-17C, IL-17D, IL-17E, and IL-17F) in mouse serum after cardiac arrest, we found that IL-17A showed the most significant changes. Figure 3 A). Furthermore, in the serum of clinical patients with myocardial infarction complicated by cardiac arrest (CA), we detected higher IL-17A expression levels compared to myocardial infarction patients without CA. Figure 3 B). These results suggest that changes in the expression of the IL-17 family, especially IL-17A, in the early post-CA / CPR period may be a key factor in regulating multi-organ function.

[0050] Figure A: Serological changes of the IL-17 family in mice during the early (3h) period after CA / CPR and sham surgery.

[0051] Figure B: Changes in serum IL-17A expression in patients with myocardial infarction complicated with cardiac arrest (CA) and patients with myocardial infarction without cardiac arrest (Non-CA).

[0052] Example 4

[0053] Early studies have investigated the effects of the anti-IL-17A monoclonal neutralizing antibody secukinumab (a human interleukin-17A antagonist purchased from Novartis, Switzerland) on improving myocardial injury and cardiac dysfunction after cardiac arrest.

[0054] After establishing the cardiac arrest model using the method described in Example 1, anti-IL-17A monoclonal neutralizing antibody secukinumab and its isotype control antibody isotype (an isotype control of human IgG1κ antibody, purchased from Selleck) were administered at the beginning of resuscitation (i.e., the early intervention stage). The administration route was via jugular vein, in combination with adrenaline. The antibody dosage was 10 mg / kg, in order to achieve early antagonism of IL-17A.

[0055] Total RNA was extracted from cardiac tissue 3 hours after resuscitation to detect changes in myocardial inflammation levels in the secukinumab and isotype groups after cardiac arrest, thus assessing myocardial injury. The Vevo2100 ultrasound imaging system was used to detect changes in cardiac systolic and diastolic function in the secukinumab and isotype groups after cardiac arrest. Specifically, long-axis and short-axis cardiac images were acquired, and the ejection fraction (EF) and fractional shortening (FS) values ​​were measured in M ​​mode to analyze cardiac systolic function; four-chamber cardiac images were acquired, and blood flow parameters were measured in PW doppler mode to analyze cardiac diastolic function. Each measurement was the average of five cardiac cycles.

[0056] The results showed that, compared with the isotype group, early antagonism of IL-17A significantly reduced the expression of chemokines in myocardial tissue and alleviated cardiomyocyte damage. Figure 4 A); significantly improves cardiac systolic function caused by cardiac arrest ( Figure 4 B) and diastolic dysfunction ( Figure 4 C).

[0057] Figure A: Early antagonism of IL-17A significantly reduced the expression of chemokines in myocardial tissue, demonstrating a significant reduction in myocardial injury.

[0058] Figure B: Echocardiography results show that early IL-17A antagonism significantly improved left ventricular EF and FS values, demonstrating a significant improvement in cardiac systolic function.

[0059] Figure C: Echocardiography results showed that early IL-17A antagonism significantly increased the left ventricular E / A ratio and reduced isovolumic relaxation time (IVRT), demonstrating a significant improvement in cardiac diastolic function.

[0060] Example 5

[0061] Early application of the anti-IL-17A monoclonal neutralizing antibody secukinumab has shown its effect on improving brain tissue damage, neurological dysfunction, and survival rate after cardiac arrest.

[0062] Using the model established and IL-17A intervention protocol described in Example 4, total RNA was extracted from brain tissue 3 hours after resuscitation to detect changes in brain inflammation levels in the secukinumab and isotype groups after cardiac arrest, and to assess brain tissue damage. Neurological function in both groups was assessed 24 hours after resuscitation. Postoperative survival was calculated up to 72 hours post-surgery, with survival observed every 3 hours during this period, and survival curves were plotted for each group.

[0063] The results showed that, compared with the isotype group, early antagonism of IL-17A significantly reduced the expression of chemokines in brain tissue and alleviated brain tissue damage. Figure 5 A); Significantly improves neurological dysfunction caused by cardiac arrest ( Figure 5 B) and improve post-recovery survival rates. Figure 5 C).

[0064] Figure A: Early antagonism of IL-17A significantly reduced the expression of chemokines in brain tissue, demonstrating a significant reduction in brain tissue damage.

[0065] Figure B: Early antagonism of IL-17A significantly improved the neurological function score 24 hours after resuscitation, demonstrating the improvement in neurological function.

[0066] Figure C: Early antagonism of IL-17A significantly improves 72-hour survival rate after cardiac arrest.

Claims

1. The use of an anti-IL-17A monoclonal antibody in the preparation of a drug for improving post-cardiac arrest syndrome, wherein the use is to improve myocardial injury, cardiac dysfunction, brain tissue injury or neurological dysfunction caused by post-cardiac arrest syndrome, wherein the anti-IL-17A monoclonal antibody is secukinumab.

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